Forty females 30–65 years of age with Fitzpatrick skin types I–VI who were diagnosed by the dermatologist investigator as possessing poor skin plumpness and hydration along with photoaging were enrolled in this single-site study to evaluate the efficacy of a facial serum. This study was conducted in compliance with the Helsinki Declaration of 1964 and its later amendments. The study was approved by the Allendale Institutional Review Board (AIRB), Old Lyme, CT). Informed consent for participation and for photograph and article publication was received from all participants. Following this, and after meeting all inclusion criteria and none of the exclusion criteria (Table 1), subjects with a lack of skin hydration and plumping and with global photoaging were enrolled. Subjects were asked to wash their face at the research center. They were provided with a headband to pull hair off their face and a black neck drape to conceal their clothing. All visible facial jewelry was removed and the subjects were instructed to close their eyes and mouth. These procedures were undertaken to anonymize the photographs.

Subjects were provided with the study facial serum (PCA Skin Hyaluronic Acid Boosting Serum, Colgate-Palmolive Co., Piscataway, NJ and PCA Skin, Scottsdale, AZ) for twice-daily use and a sunscreen (Neutrogena Clear Face Broad Spectrum SPF 55, Johnson & Johnson, Skillman, NJ) for use as needed during the study. The use of facial sunscreen was required by the IRB and it was used by all subjects, as documented in the subject diaries. Subjects used their own self-selected cleanser and cosmetics, but these products were stable for 30 days prior to study entry and throughout the study. A compliance diary was provided to record the use of the study serum.

The dermatologist investigator evaluated smoothness, plumping, hydration, fine lines/wrinkles, and global appearance issues. The following 5-point ordinal scale was used: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe. Subjects assessed product tolerability in terms of stinging, itching, and burning. The following 5-point ordinal scale was used: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe. Corneometry (Dermalab Combo Pin Probe, Cortex Technologies, Hadsund, Denmark) of the left cheek was conducted for all subjects in triplicate after they had acclimated to the study environment for 20–30 min.

VISIA CR4.3 (Canfield Scientific, Parsippany, NJ) photography of the front, right, and left face with visible light was conducted for a subset of 15 subjects. The faces of these 15 subjects were then swabbed on the right cheek for HA levels after photos had been captured at each study visit so as not to interfere with images. Swabs were collected on clean skin, as each subject was asked to wash their face with their self-selected cleanser at the research center prior to all study assessments during their afternoon study visits (the last product application was completed in the morning of the study visit at weeks 2, 4, and 6). The swabbing technique is often used to collect skin surface microbiome samples. HydraFlock (25-3606-H, Puritan Medical Products Company LLC, Guiford, ME) was first wetted by dipping it into sterile phosphate-buffered saline (PBS). A skin surface sample was collected by covering a surface area of 2 inches by 2 inches with a swab while rotating and moving the swab. Swabs collected throughout the study were stored at – 80 °C until processing. Each swab was soaked and shaken in 1 mL of PBS with protease inhibitor cocktails for 1 h at room temperature. Freezing/thawing of the extracts to/from – 80 °C was limited to three times within 2 weeks which did not impact the ELISA results. The resulting extracts were used in interleukin-1a (IL-1a, SLA50, Human IL-1 Alpha/IL-1F1 Quantikine ELISA Kit, R&D Systems Minneapolis, MN), hyaluronan (DHYAL0, Hyaluronan Quantikine ELISA Kit, R&D Systems, Minneapolis, MN), and filaggrin (CSB-EL008712HU, Cusabio, Houston, TX) enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer’s protocols.

During the baseline visit only, all subjects applied the study product and sat for 10–15 min at the end of their study visit after all assessments were completed. Investigator assessments were completed for immediate plumping and hydration. Postapplication corneometry of the left cheek was conducted for all subjects in triplicate. Subjects returned for the same assessments at weeks 2, 4, and 6.

Full-thickness human skin equivalents from MatTek (EpiDermFT, EFT-400, Ashland, MA) were used to evaluate whether the study HA serum product could increase hyaluronan expression. After equilibration overnight after receiving EFT-400, 10 μL of the product were applied on top of the skin equivalents for further incubation at 37 °C with 5% CO2. Triplicates were used for untreated samples and duplicates for the serum-treated samples. After 24 h of treatment, the medium was collected for IL-1a release. Tissues were rinsed with PBS three times and collected for HA expression. The epidermis and the dermis were separated from tissues for protein extraction using a TissueLyser II (85300, Qiagen, Carlsbad, CA) and RIPA lysis buffer (R0278, Sigma-Aldrich, St. Louis, MO). The protein concentration was determined using a Micro BCA Protein Assay Kit (23235, Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol. IL-1a release and hyaluronan expression were determined using ELISAs (SLA50, DHYAL0, R&D Systems) according to the manufacturer’s protocols. For the hyaluronan ELISA, protein lysates were diluted 1000-fold (epidermis) and 10,000-fold (dermis) and normalized to the protein concentration.

Levels of HA and other active ingredients in the PCA Skin Hyaluronic Acid Boosting Serum are kept proprietary. The serum contains HA as well as HA Pro Complex containing disodium acetyl glucosamine phosphate, hydrolyzed yeast extract, polyglucuronic acid, and sodium carrageenan.